We will generate translationally essential information on the process of ?-cell differentiation from progenitor cells, targeting a major outstanding issue: what stimuli and culture conditions to employ to produce mature ?-cells in vitro. Our team will focus solely on the tissue source of all ?-cell progenitors: the trunk domain epithelium of mid-pancreas organogenesis. The spatiotemporally orchestrated signaling networks and cellular division/migration processes that control ?-cell birth/maturation are very poorly defined. But, cell-autonomous and non-autonomous programs that give rise to mature b cells must work within the constraints of a defined epithelial structure, with its tightly linked morphogenetic program that produces the large numbers of budded, mature islets. We will perform a high-resolution structural and cell biological analysis of endocrine progenitor locations, cell-cell organization and signaling, and their behavior in yielding differentiating ?-cell progeny or remaining as cycling endocrine-biased cells within the epithelium. We will: (1) Define how the epithelial structure provides signaling niches that control the location, number and behavior of endocrine-biased progenitors. (2) Define how asymmetric division yields pro-endocrine precursors and endocrine-committed progeny. (3) Characterize the directional delamination of pro-endocrine cells and their progression to ?-cell or other endocrine types. (4) Localize intermediates marked by cell-instructive factors, and determine cell-autonomous and cell-non-autonomous effects. (5) Use mutational analysis and lineage tracing to detail the intraepithelial organization and signaling processes involved. (6) Perform gene expression profiling of selected cell populations in normal and perturbed situations to provide an unprecedented understanding of the gene regulatory networks involved, plus new insights into markers of critical transitional cell states. (7) Map new monoclonal antibodies from the BCBC cell-surface-marker project onto this framework, providing new tools for identifying/sorting progenitor-progeny states in differentiating hESC. (8) Test directly the functional information emerging from our studies as ?-cell formation/maturation stimuli on human ES cell (hESC) differentiating in vitro. Such a spatiotemporally resolved 'normal differentiation framework'will shed light on deficiencies in hESC differentiation protocols. In vitro hESC-derived ?-cells are immature and low in number, while partly differentiated, propancreatic cell clusters produce functional ?-cells on maturation in mice: we hypothesize that the in vitro deficit reflects an inability to build proper communication and progenitor qualities. We designed our project to integrate with and complement studies of other BCBC teams on hESC differentiation and endocrine progenitor identification. The team's pedigree in lineage analysis, cell biology and progenitor control, longevity and strong intra-BCBC interactions, led to our sharply defined common interest in epithelial endocrine progenitors. Novel tools, reagents, data sets will be shared with all BCBC members.